| Backgroud:Hand, foot and mouth disease (HFMD) is a common contagious disease among children, which always characterized with fever, oral ulcerations and vesicular rash (exanthema) on the palms, soles and/or buttocks. With an increasing incidence, HFMD is presenting a trend of seasonal epidemic with the sporadic cases in a whole year recently. Besides EV-71 and CV-A16, CV-A10 has been associated with increasingly occurred sporadic HFMD cases and outbreak events globally, compared with other HEV.Most diseases associated with CV-A10 were wild and self-limiting, but there also have severe and death cases been reported in domestic and overseas. Although CV-A10 occupied certain proportion in HFMD pathogenic spectrum, the research about its component ratio in HFMD, gene mutation in VP1 and geographical distributionin China are still not clear. This study, we focus on analyzing the gene mutation in VP1 region of CV-A10 strains that isolated in HFMD in China. We also analysed the recombination in 3D region of CV-A10 strains.what aim to explore the molecular epidemiology of CV-A10 in China,Objective:In order to formulate reasonable treatment measures and provide scientific basis for the prevention and control of HFMD, this study was to explore the dynamic circulation and genetic characteristics of CV-A10 with HFMD in China. Fifty-seven CV-A10 strains were isolated from children with HFMD during 2009 to 2012 in nine provinces of China; the gene variants change rules in the VP1 region and the genetic recombination analysis of 3D region were also analysised.Meathods:All the isolates were from prefectural Centers for Diseases Control and Prevention and collected by National Laboratory since 2009. Total RNA were extracted from the isolate sand amplified by RT-PCR with the universal primers of enteroviruses after the rRT-PCR. All the PCR products then were sequenced directly, and the sequences were spliced and assembled by Sequencher 5.0 (GeneCode, Ann Arbor, Michigan, USA). Assembled sequences were identified by BLAST, obtained online from the National Center for Biotechnology Information (NCBI) were collected. Virus strain showing>75% nucleotide sequence identity to CV-A10 was assigned to that serotype.Entire VP1 gene and 3D region of CV-A10 strain were amplified by reverse transcription-PCR (RT-PCR) with specific primers designed. Phylogenetic analysis with all of available sequences downloaded from GenBank based on entire VP1 sequences. Two epresentative strains that rooting in genetype B and C were selected, whose sequences of 3D region with all the HEV-A strains and the closest relative sequences by the Blasting were analysised by MEGA.Reasults:This study Phylogenetic analysis with all of available sequences downloaded from GenBank based on entire VP1 sequences showed that CV-A10 strains were divided into four genotypes. Almost all the sequence from China, forming genotype C, shared 93.93%-100% and 96.84%-100% identities in nucleotides and amino acids. Genotype B include one isolate from Hunan province of China in 2009, two sequences from Shenzhen in 2009, and five sequences from Shandong, China:three in 2008, each one in 2004 and 2006, respectively. Homologous analysis revealed that 78.90% to 84.52% nucleotide identities between genotype B with the other genotypes, 2.41% to 9.29% divergence within genotype.1. Genotype C of Chinese CV-A10 strains, which nucleotide homology of 93.93%? 93.93%, amino acid homology was 96.84%?100%;This 5 years strains exist obvious s relationship with the evolution of the genetic variation changes,2011 ? 2012 strains of cluster 1, gathered at the top of the tree; and 2008?2010(cluster 2), at the end of the tree.The nucleotide differences between two cluters was 3.6%?6.2%;2. The strain of Genotype B isolated in China from 2004 to 2009, including one strain from Hunan province in 2009,5 strains of Shandong province from 2004 to 2008 and two strains of Shenzhen in 2009. The nucleotide homology between these sequences were 90.6?97.5%, and 97.8%?100% in amino acid; Its nucleotide differences with other genotypes was 15.48%?21.9%, with the amino acid differences of 4.7% to 4.7%.3. According to the 3D sequences, we could infer that there have been a significant restructuring in CV-A10 strains, and the different genotypes had two different ways of restructuring. The Genotype C of CV-A10 strain in this study isolated in 2012(CQ12-6) show a high homologies with the prototype of CV-A5, and the sequence homo logy between them was 81.7%; The Genotype B of CV-A10 strain in this study isolated in 2009 of China(HuN09-22)have a high homologies with one CV-A6 strain isolated from Taiwan's (JQ946050), the nucleotide homology was 94.3%. Besides, three prtotypes of CV-A4?CV-A14 and CV-A16 were in the same evolutionary line, whose homologies were83.6%,83.0% and 84.1%, respectively.4. According to the China disease prevention information system, the enterovirus of non-EV-A71 and non-CV-A16 has a gradually rising proportion in laboratory test cases in Sichuan province duiring 2011?2013, and the proportion was 15%, 33%,48%,respectively. The identification for separation of other enterovirus showed that CV-A6 (51%) and CV-A10(10%) were the main pathogenic agents.Conclusion:1. There are two genotypes of CV-A10, genotype B and C, circulated in our country, and its VP1 region showed an obvious s dynamic evolutionary relationships; Besides, the two genotypes of Chinese CV-A10 strain in 3D region revealed that different genotype restructured evolution in different ways;2. In recent three years CV-A6 and CV-A10 that caused HFMD has increasing trend in the enterovirus.3. This study formulate reasonable treatment measures and provide scientific basis for the prevention and control of HFMD in China. |
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